A long term goal is to gain an overall understanding of the evolution, gene organization, and regulation of the tRNA multigene family in Escherichia coli. Specific tRNA genes and regulatory DNA will be utilized for detailed studies on gene expression in vivo and in vitro. We are focusing our efforts on the tRNA-leu (leuV) operon and are attempting to elucidate the detailed mechanisms for growth rate dependent regulation and stringent control. We shall compare these results with that obtained using four other E. coli tRNA promoters in order to determine common or unique properties of regulatory DNA. Chemical synthesis and in vitro mutagenesis will be the major approaches employed to identify specific sequences necessary and sufficient for promoter functions. We have shown that some tRNA genes may exhibit a form of feedback control recently shown to be operative for rRNA operons, and will investigate the molecular basis for this phenomenon using appropriate mutant promoters and host strains. In addition, we have shown that the leuV promoter is activated by upstream sequences in DNA; we will investigate this novel parameter in vivo and in vitro using modified promoters in an attempt to determine the molecular basis of activation. This work impinges on several major unanswered questions such as: How are all the tRNA genes of a cell coordinately expressed to ensure accurate protein synthesis? What are the general structural features of the genes? What factors control initiation and termination of transcription here? Ultimately, these studies should permit the engineering of modified genes which will be useful in understanding how tRNA works in the cell.